Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sites
Biotin labeling in combination with mass spectrometry has been widely applied in large-scale biological studies, such as determination of protein partners, protein subcellular localization, and protein post-translational modifications. Previous studies have shown that immunoaffinity enrichment is a...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-07-01
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| Series: | Biochemistry and Biophysics Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S240558082400075X |
| _version_ | 1797200789271216128 |
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| author | Yiying Zhu |
| author_facet | Yiying Zhu |
| author_sort | Yiying Zhu |
| collection | DOAJ |
| description | Biotin labeling in combination with mass spectrometry has been widely applied in large-scale biological studies, such as determination of protein partners, protein subcellular localization, and protein post-translational modifications. Previous studies have shown that immunoaffinity enrichment is a better method than streptavidin/avidin purification for site-specific studies of biotinylated molecules. In this study, we made a crucial improvement to the elution phase of the immunoaffinity enrichment step for biotinylated peptides, which involves the addition of a highly organic solvent, and developed a monoclonal anti-biotin antibody that improved the identification number for biotinylated peptides. We then demonstrated its application in the characterization of protein interaction sites for the β2 adrenergic receptor (β2AR) by proximity labeling in living cells. Our research provides an improved and reproducible immunoaffinity enrichment method for site-specific biotin-related research. |
| first_indexed | 2024-04-24T07:37:14Z |
| format | Article |
| id | doaj.art-428e4a2078e044378c75c0a498a2e6d2 |
| institution | Directory Open Access Journal |
| issn | 2405-5808 |
| language | English |
| last_indexed | 2024-04-24T07:37:14Z |
| publishDate | 2024-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biochemistry and Biophysics Reports |
| spelling | doaj.art-428e4a2078e044378c75c0a498a2e6d22024-04-20T04:17:33ZengElsevierBiochemistry and Biophysics Reports2405-58082024-07-0138101711Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sitesYiying Zhu0Tsinghua University and Cell Signaling Technology Inc, Chemistry Department, Tsinghua University, Beijing, 100084, ChinaBiotin labeling in combination with mass spectrometry has been widely applied in large-scale biological studies, such as determination of protein partners, protein subcellular localization, and protein post-translational modifications. Previous studies have shown that immunoaffinity enrichment is a better method than streptavidin/avidin purification for site-specific studies of biotinylated molecules. In this study, we made a crucial improvement to the elution phase of the immunoaffinity enrichment step for biotinylated peptides, which involves the addition of a highly organic solvent, and developed a monoclonal anti-biotin antibody that improved the identification number for biotinylated peptides. We then demonstrated its application in the characterization of protein interaction sites for the β2 adrenergic receptor (β2AR) by proximity labeling in living cells. Our research provides an improved and reproducible immunoaffinity enrichment method for site-specific biotin-related research.http://www.sciencedirect.com/science/article/pii/S240558082400075XBiotinylationImmuno-affinity enrichmentSite-specific analysis |
| spellingShingle | Yiying Zhu Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sites Biochemistry and Biophysics Reports Biotinylation Immuno-affinity enrichment Site-specific analysis |
| title | Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sites |
| title_full | Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sites |
| title_fullStr | Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sites |
| title_full_unstemmed | Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sites |
| title_short | Improved elution strategy and new monoclonal anti-biotin antibody for LC-MS/MS characterization of protein biotinylation sites |
| title_sort | improved elution strategy and new monoclonal anti biotin antibody for lc ms ms characterization of protein biotinylation sites |
| topic | Biotinylation Immuno-affinity enrichment Site-specific analysis |
| url | http://www.sciencedirect.com/science/article/pii/S240558082400075X |
| work_keys_str_mv | AT yiyingzhu improvedelutionstrategyandnewmonoclonalantibiotinantibodyforlcmsmscharacterizationofproteinbiotinylationsites |